03/09f Diagnostic Virology

Question 1

Is there such thing as a ‘broad-spectrum’ antiviral?

Answer 1

NO - antivirals are specific to certain viruses

Question 2

Why do we need specific diagnoses for viruses?

Answer 2

To treat infections with specific antiviral chemotherapy
To institute appropriate control measures
To decrease inappropriate therapy and unnecessary hospitalization
For education and awareness of viral disease

Question 3

What are the strategies to diagnose viral infections in the lab? Name four

Answer 3

1) Viral isolation in cell culture systems
2) Antigen detection of proteins unique to specific viruses
3) Nucleic acid detection through PCR
4) Detection of immune response in the host

Question 4

Why is viral isolation a nonspecific means of detecting virus?

Answer 4

ANY virus might result from culture isolation, including one that was not in your differential

Question 5

What information must be used in conjunction with viral cell culture in order to make a preliminary diagnosis?

Answer 5

Clinical history
Source of specimen
Timing and characteristics of cytopathic effects
Host range of virus in vitro

Question 6

What is a shell vial?

Answer 6

A vial that contains an enhanced environment to encourage fast virus growth

Question 7

What are the advantages of viral isolation? List three

Answer 7

1) Gold standard of diagnosis for viruses that will growth in cell culture
2) Permits detection of unexpected viruses
3) Permits the performance of additional assays such as identification and speciation, antiviral susceptibility, and PCR/sequencing

Question 8

What are the disadvantages of viral isolation? List four

Answer 8

1) Maintenance of cell culture is expensive, labor-intensive, and slow
2) Not available in all labs
3) Some viruses only grow in certain cell culture systems
4) Some viruses won’t grow in culture at all

Question 9

What is viral antigen detection?

Answer 9

Method that uses tagged monoclonal antibodies to identify specific viral epitopes
Must have an idea of what virus it might be

Question 10

What are the advantages of antigen detection?

Answer 10

1) Rapid (1-2 hours)
2) Does not require the presence of infectious or viable virus
3) Does not require cold-chain specimen transport
4) Amenable to batch testing
5) Able to assess specimen quality

Question 11

What are the disadvantages of antigen detection?

Answer 11

1) Less sensitive than other methods because there is no amplification of the sample
2) Requires specific reagents, some of which can be carcinogenic
3) Some specimens are not suitable for antigen detection (result in nonspecific reactions)
4) Not applicable to all viruses

Question 12

What is fluorescent antibody staining?

Answer 12

Method that uses a monoclonal antibody specific to a viral epitope, and a fluorescent marker
Widely used for viral antigen detection

Question 13

What are the two procedures possible for fluorescent antibody staining?

Answer 13

Direct - antiviral antibody is conjugated with a fluorescent marker
Indirect - uses fluorescently-tagged secondary antibody

Question 14

What are the advantages of fluorescent antibody techniques?

Answer 14

Direct - simple, fast

Indirect - more sensitive

Question 15

What are the disadvantages of fluorescent antibody techniques?

Answer 15

Direct - requires fluorescent conjugation to each antiviral antibody, which is expensive
Indirect - more time-consuming
Both are subjective and complex tests

Question 16

What is an enzyme immunoassay?

Answer 16

Method that uses a ‘capture’ antibody that is fixed to a solid surface and binds the antigen of interest
Antigen is directly or indirectly detected with a secondary antibody
Substrate produces a quantifiable color change (read with a spectrophotometer)

Question 17

What are the advantages to EIAs?

Answer 17

1) Rapid
2) Can be batched and automated for large volumes of specimens
3) Can also be performed individually on a STAT basis
4) Can be applied to diverse type of specimens
5) Does not require intact live virus

Question 18

What are the disadvantages of EIAs?

Answer 18

1) Less sensitive than culture
2) Most monoclonal antibody has low affinity - can be washed away
3) Requires very specific reagents
4) Not applicable to all viruses

Question 19

What are three methods of nucleic acid detection of viruses?

Answer 19

1) Direct hybridization
2) Target (template) amplification - PCR, used most often
3) Signal amplification

Question 20

What is direct hybridization? What specific assays rely on this technique?

Answer 20

Direct detection of viral nucleic acid without amplification, using a nucleic acid probe
Assays include dot blot, liquid hybridization, and Southern blot
Lack sensitivity - best for specimens with a high titer of target protein (like Hepatitis B)

Question 21

What is target amplification?

Answer 21

PCR

Question 22

What are the advantages of PCR?

Answer 22

Highly sensitive

Rapid

Question 23

What are the disadvantages of PCR?

Answer 23

Too sensitive for some applications (latent viruses)
Expensive
Reaction can be inhibited by a variety of substances

Question 24

What is signal amplification?

Answer 24

Method in which nucleic acid is hybridized to a probe (nucleic acid is NOT amplified)
Signal is amplified by binding many detector molecules to each target-probe hybrid
Example - hybrid capture assay

Question 25

What are the advantages of signal amplification?

Answer 25

Simpler than target amplification

Not susceptible to carryover contamination (unlike PCR)

Question 26

What are the disadvantages of signal amplification?

Answer 26

Not as sensitive as PCR

Question 27

How do you obtain serologic evidence of an acute infection?

Answer 27

Look for seroconversion in serial specimens - rising or falling titer of virus-specific antibodies from acute to convalescent phase
Presence of virus-specific IgM in an acute phase specimen can indicate infection

Question 28

In what types of viral infections do you want to test CSF in addition to serum?

Answer 28

Rabies - virus-specific antibody in CSF is diagnostic

Encephalitis viruses - virus-specific IgM is diagnostic

Question 29

What are the advantages of serological tests?

Answer 29

Useful for viruses that can’t be cultured
For certain viruses (that usually cause chronic infection), the presence of antibodies is diagnostic
Determines patient’s immune status to specific viruses

Question 30

What are the disadvantages of serological tests?

Answer 30

May take a long time (need both acute and convalescent samples)
May not be accurate for patients who have received a number of blood transfusions
Some viral infections may react non-specifically (false positive)